Succinic acid is a dicarboxylic acid comprised of four carbon atoms and can be used in various industrial applications. Succinic acid can be used as a precursor of industrially important chemicals, including adipic acid, 1,4-butanediol, ethylenediamine disuccinate, itaconic acid, γ-butyrolactone, γ-aminobutyric acid, and tetrahydrofuran, and the global market size of succinic acid, including precursors thereof, is estimated to be about 15 billion dollars (McKinlay et al., Appl. Microbiol. Biotechnol., 76:727, 2007). With recent interest in environmentally sustainable development and due to decreasing oil reserves and the resulting price fluctuations, global studies on the production of bio-based succinic acid have been conducted over past decades (McKinlay et al., Appl. Microbiol. Biotechnol. 76:727, 2007; Song et al., Enzyme Microbial Technol., 39:352, 2006; Jantama et al., Biotechnol. Bioeng., 99:1140, 2008). However, any kind of strain developed to date did not make it possible to maximize the productivity and yield of succinic acid while minimizing byproduct production. When the productivity was high, the efficiency was low, and conversely, when the efficiency was high, the productivity was low. Further, when the productivity was high, large amounts of byproducts were also produced. Thus, an ideal strain that can increase productivity and yield while producing only succinic acid has not yet been developed (Jantama et al., Biotechnol. Bioeng., 99:1140, 2008).
Sucrose has a price of about ¼ of the price of glucose that is generally used to produce succinic acid by microbial fermentation. Also, with a rapid increase in global biodiesel production, glycerol is being produced as a byproduct, and thus the price thereof is decreasing due to excessive supply and an appropriate method for treating glycerol is required. Accordingly, the price of glycerol is very low and continues to decrease (Miller-Klein Associates, October 2006).
Meanwhile, most microorganisms preferentially utilize preferred carbon sources from mixtures of different carbon sources. To make it possible, most microorganisms have a catabolite repression mechanism that inhibits the utilization of non-preferred carbon sources when preferred carbon sources are available (Gorke et al., Nature Reviews, 6:613, 2008). With respect to the preferential utilization of carbon sources regulated by a catabolite repression mechanism, it is well known that E. coli shows diauxic growth in the presence of both glucose and lactose, as reported by Monod et al. in 1942. Herein, the preferred carbon source is glucose, and thus E. coli shows a diauxic growth curve in which the non-preferred carbon source lactose starts to be consumed after a short lag phase after glucose has been completely consumed. Also, because of this catabolite repression mechanism, it is very difficult for general Mannheimia strains to utilize sucrose and glycerol at the same time. Nevertheless, the utilization of glycerol as a carbon source offers many advantages. Glycerol is highly reduced and when it is used as a carbon source, reducing equivalents (NADH, NADPH, FADH2, etc.) are produced in an amount two times larger than sugars such as glucose, xylose and sucrose during the production of the intermediate phosphoenolpyruvate (PEP). Thus, glycerol is an attractive carbon source for the production of reducing chemicals (Yazdani et al., Curr. Opin. Biotechnol., 18:213, 2007). However, in many cases, the rate of growth of cells by the utilization of glycerol under anaerobic conditions is slower than that by the utilization of other sugars, and thus the utilization of glycerol as a single carbon source is advantageous in terms of reducing power, but has a limitation in increasing the productivity of a desired biological product because it shows slow growth rate.
To overcome this limitation, if the utilization rate of glycerol can be increased while utilizing sugars such as sucrose, which have a higher utilization rate than glycerol and enable cell growth at a higher level and rate, cells can be growth at a high rate while using the advantage of the high reducing power of glycerol, and thus reducing compounds, particularly succinic acid, can be effectively produced.
Accordingly, the present inventors have made extensive efforts to develop a method of producing high-purity succinic acid with high efficiency by using inexpensive sucrose and glycerol simultaneously, and as a result, have found that, when a succinic acid-producing mutant microorganism, obtained by deleting a fructose phosphotransferase-encoding gene from a succinic acid-producing microorganism or introducing a glycerol kinase-encoding gene into the microorganism, is cultured, the catabolite repression mechanism in the mutant microorganism is relieved so that the mutant microorganism can produce succinic acid using sucrose and glycerol simultaneously, minimize the production of byproducts, and produce homo-succinic acid with high efficiency and a very high productivity which could not be attained in conventional methods, thereby completing the present invention.